Transmitted by mosquitoes, dengue is among the most important reemerging infectious diseases with an estimated 50 million to 100 million annual cases, 500,000 hospitalizations, and 22,000 deaths. Breaking the link between the mosquito vector and the dengue virus is the best option for protecting people. The endosymbiotic bacterium, Wolbachia, has long been promoted as potential vehicles for introducing disease- resistant genes into mosquitoes, making them refractory to the pathogens they carry. Pathogens are known to interact with Wolbachia and other symbiotic bacteria in several insect species. Given the large overlap in tissue distribution and intracellular localization of Wolbachia and dengue virus in mosquitoes, it is imperative that we characterize their interaction and determine the influence of Wolbachia on dengue transmission. Our long-term goal is to develop Wolbachia-based control strategies to block dengue virus transmission in mosquitoes. The objective of this proposal is to identify factors that enable Wolbachia-based population replacement to succeed in a way that reduces vector capacity to dengue virus. Our central hypotheses are that interactions between Wolbachia and dengue virus affect both the vector capacity of Ae. aegypti to dengue virus and the ability of Wolbachia to serve as a transgene driver. We formulated these hypotheses based on preliminary data that showed: 1) Wolbachia and dengue virus induce a host innate immune response and 2) the host immune response was correlated with the elimination of Wolbachia and suppression of dengue virus. The project has the five specific aims: 1). Initiate Wolbachia stock in Ae. aegypti. These stocks will be used on studies of strain- specific Wolbachia interactions with Ae. aegypti and dengue virus. 2). Identify molecular factors that determine the interactions between Wolbachia and dengue virus. We will design a novel dual-genome microarray, and utilize it to monitor gene expression changes in both Ae. aegypti and Wolbachia simultaneously in response to dengue infection. 3). Identify changes in dengue viral dynamics within Ae. aegypti due to Wolbachia infection. We will investigate the impact of Wolbachia on the oral susceptibility of mosquitoes to dengue virus, virus dissemination and transmission, and the potential of Wolbachia to select for more invasive dengue strains. 4). Identify Wolbachia-host interactions that are impaired by dengue infection. We will assess the impact of dengue virus on Wolbachia infection level, cytoplasmic incompatibility and maternal transmissibility. 5). Evaluate the mosquito fitness costs associated with dual infections of dengue virus and Wolbachia. We will focus on the fitness cost on the mosquito longevity, fecundity, and blood-feeding behavior. We anticipate this knowledge will improve genetic technology and its application in reducing vector capacity and blocking the transmission of dengue fever, thus, lead to better understand, treat, and prevent dengue fever.